In previous reports we have described the use of microarray and proteomic techniques together with our polyamine-requiring mutants of Saccharomyces cerevisiae to find which gene and proteins are upregulated or downregulated by spermidine supplementation to polyamine -deficient cultures. We found changes in a very large number of genes and proteins. Therefore we have now focused on the earliest changes after spermidine supplementation, and preliminary studies indicate a preponderance of genes involved in carbohydrate metabolism. Forty five genes are induced by spermidine at an early time points (30 min), among those 25% are related to carbohydrate metabolism in yeast (such as GCY1, GLK1, GPH1, GSY2, HXK1, PGM2, SOL4, TDH1, TPS2, TSL1, UGP1). We are also carrying out microarray studies with our Escherichia coli mutants that lack all of the enzymes involved in polyamine-biosynthesis.. For this purpose we have developed the use of a chemostat since we feel that it is very important not to have the results complicated by changes in the growth rates after spermidine addition. Lysine and arginine auxotrophs have also being constructed to permit the use of the SILAC technique for proteomic studies. Our current studies in E. coli have been particularly concerned with the physiologic function of glutathionylspermidine. As we have previously shown, all of the spermidine of the E. coli cells and a very large percentage of the intracellular glutathione are converted to glutathionylspermidine at the end of the logarithmic growth. In the current work we have concentrated on the formation and function of this derivative in logarithmically growing cultures. Nothing is known about the function of glutathionylspermidine in E. coli, although it has been shown to be of critical importance in trypanosomes. A mutant of E. coli is available that lacks the gene for glutathionylspermidine synthetase/amidase (gsp). We have compared the phenotypic effects of the gsp gene deletion in E. coli with a wild type strain in various growth conditions (air, 95% oxygen, temperature and copper toxicity) and found no difference between the mutant and the wild type. In our current work we have carried out detailed microarray studies, comparing the wild type and gsp mutant strain (with the help of the NIDDK Microarray facility). We have standardized a technique to remove rRNA from E. coli total RNA and used the enriched mRNA for this comparison. The recent results show a large effect of deletion of gsp gene. Around 160 genes were upregulated more than 2-fold in mutant cells as compared to wild type cells, which include genes for purine and pyrimidine nucleotide biosynthesis, arginine and putrescine metabolism. Approximately 120 genes were down-regulated more than 2-fold in gsp mutant. The genes for metalochaperone, molybdenum, copper, zinc and silver ion transport were down-regulated together with grxA (glutaredoxin 1, a redox coenzyme for ribonucleotide reductase), and nitrite transporter nirC. Detailed proteomic studies have also been carried out (in collaboration with Dr. Eric Anderson of the NIDDK Mass Spectrometry section) comparing the protein composition of the two strains using the SILAC technique. For this purpose we have constructed strains containing deletions in the lysA and argA genes. Very recently we have obtained data from this mass spectroscopy analysis, and found 8-10 peptides are upregulated or downregulated in the mutant.